Seroprevalence of Leptospirosis in Cattle in Papua New Guinea

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1 Seroprevalence of Leptospirosis in Cattle in Papua New Guinea Abstract Wai in, P 1, Robertson, I. 1, Fenwick, S. 1, Smythe, L. 2 and Reid S School of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, WA WHO/FAO/OIE Collaborating Centre for Reference and Research on Leptospirosis, P.O. Box 594, Archerfield, Queensland 4108, Australia Leptospirosis is an economically important bacterial infection in cattle that causes abortion, stillbirth, infertility and reduced milk production. It is responsible for reducing livestock production and the importance of this disease lies in it being an infectious disease of livestock and rodents which has the potential to infect humans. The extent of leptospirosis infection in the livestock population in Papua New Guinea has not been studied and this paper reports on the findings of a study carried out in March to April 2004 on six cattle farms. Sera from 1300 female cattle in three age groups (<2, 2-5 and >5 years) from six farms were collected and tested for leptospiral antibodies using the microscopic agglutination test. In addition, 79 matched blood and kidneys were collected from the central abattoir, part of the kidney was cultured in EMJH media. The overall seroprevalence of leptospiral serovars detected were Hardjo (53.7%, 95% CI: ), Szwajizak (30.2%, 95% CI: ), Tarassovi (15.8%, 95% CI: ), Medanensis (12.8%, 95% CI: ), Kremastos (6.6%, 95% CI: %), Pomona (8.4%, 95% CI: ). Leptospires was isolated from two kidneys and typed to be L. borgpetersenii serovar Hardjo. In conclusion the main leptospiral serovar infecting cattle is Hardjo and cattle are a potential zoonotic reservoir for serovars Hardjo, Tarassovi and Pomona in Papua New Guinea. Keywords: Leptospirosis; cattle; Hardjo; seroprevalence; Papua New Guinea

2 Introduction Leptospires belonging to Leptospira serovar Hardjo are the most common cause of leptospirosis in cattle (Ellis, et al. 1978; Thiermann, 1983). Leptospirosis is a major cause of economic loss to the livestock industry as a result of reduced fertility (Hathaway et al. 1982) and disease in people associated with livestock (Blackmore, et al. 1982; Smythe, et al. 2000). There was concern raised by the PNG cattlemen association of reduced animal production and leptospirosis suspected. Reliable estimate of the prevalence of leptospira infection in cattle in Papua New Guinea is not available. The aims of this study were to determine the extent of leptospiral infection and which is the main serovar infecting the cattle population and to the role of cattle as a reservoir for zoonotic leptospiral serovars. Materials and Methods Location Five cattle farms were located in Lae and the sixth farm is in Kimbe in the West New Britain province in Papua New Guinea were visited in March to April of 2004 (Figure 1). Lae is situated 6 0 south of the equator on the northeast coast of Papua New Guinea. Kimbe is an island east of the mainland and is 5 0 to the south and to the east of the equator. The climate is divided into two distinct seasons wet and dry, and average annual rainfall is 4600mm and daily temperatures vary from 22 to 32 0 C. Farms are all commercial enterprises and represent approximately 90% of the total cattle population with a herd size varying from 200 to 10,500. The central abattoir in Lae was visited and blood and kidneys were collected from cattle. B C E D A Lae F Kimbe Figure 1 Map of Papua New Guinea showing the farms surveyed. Collection of samples 1300 blood samples were collected from female cattle from the farms. Farm A, 369 blood of which 126 were <2 years, 122 were 2-5 years and 121 were >5 years. Farm B, 224 blood of which 71 were <2 years, 71 were 2-5 years and 82 were >5 years. Farm C, 172 blood of which 47 were <2 years, 54 were 2-5 years and 71 were >5 years. Farm D, 134 blood of which 67 were <2 years, 45 were 2-5 years and 22 were >5 years. Farm E, 128 blood of which 45 were <2 years, 21 were 2-5 years and 62 were >5years. Farm F, 273 blood of which 84 were <2 years, 83 were 2-5 years and 106 were >5 years. In addition 79 bloods with matched kidneys were collected from the central abattoir in Lae. Blood were

3 left overnight to clot at 4 0 C before centrifugation. Serum was then removed, labelled and stored at C. Culture The Ellinghausen-McCullough-Johnson-Harris (EMJH) semi-solid medium containing 5-fluorouracil was prepared by the WHO/FAO/OIE Collaborating Centre of Reference and Research on Leptospirosis in Brisbane and was shipped to Papua New Guinea for the purpose of this work. Kidneys from cattle slaughtered at the abattoir were collected and processed for culture. A 1-2 mm 3 from the medullary region of the kidney was removed and placed into the EMJH semi-solid agar. The EMJH media was incubated at 28 0 C. Cultures were sent to the WHO/FAO/OIE Collaborating Centre of Reference and Research on Leptospirosis in Brisbane for identification and typing. Microscopic agglutination test (MAT) All sera were tested against a reference panel of 21 leptospiral serovars: Pomona, Hardjo, Tarassovi, Grippotyphosa, Celledoni, Copenhageni, Australis, Zanoni, Robinsoni, Canicola, Kremastos, Szwajizak, Medanensis, Bulgarica, Cynopteri, Ballum, Bataviae, Djasiman, Javanica, Panama and Shermani using the microscopic agglutination test (MAT). Sera with antibody titre of >50 were considered positive. Analysis Data were entered in Microsoft Excel and the statistical package SPSS version was used to determine seroprevalence and 95% confidence intervals calculated using the excel template provided by Assoc. Prof. Dr Ian Robertson. Associations of seroprevalence with age were assessed with a chisquare of independence. Results Agglutinins to 15 serovars were detected sera and the most prevalent serovar was Hardjo (53.7%, 95% CI: 51.1, 56.4). Other serovars detected were Szwajizak (30.2%, 95% CI: 27.7, 32.6), Tarassovi (15.5%, 95% CI: 13.6, 17.4), Medanensis (12.8%, 95% CI: 11, 14.5), Pomona (8.4%, 95% CI: 6.9, 9.9), Kremastos (6.6%, 95% CI: 5.3, 7.9). Antibodies to serovars Ballum, Bataviae, Shermani, Djasiman, Australis, Zanoni, Panama, Robinsoni, and Celledoni were also detected but each at a level <1%. In the 3 age groups serovar Hardjo was dominant (<2years 26.8%, 95% CI: 22.7, 30.8; 2-5 years, 62.6%, 95% CI: 58.1, 67.2; >5 years 71%, 95% CI: 67, 75.1). The seroprevalence of Hardjo in the <2 years is significantly lower than that of older cattle (p < 0.05). Antibodies to serovars Hardjo, Tarassovi and Pomona are detected in all farms and Hardjo is prevalent with > 30% (Figure 1). Cattle in all ages were actively infected with serovar Hardjo (Figure 2). The seroprevalence of heifers (<2 years) acquiring infection is significantly lower (p < 0.05) compared to the older cattle. An average of 1 in every 5 heifers will acquire active infection with serovar Hardjo. Leptospires was isolated from two kidneys and typed to be L. borgpetersenii serovar Hardjo.

4 Figure 1 Distribution of serovars Hardjo, Tarassovi and Pomona on the farms. MAT titre > 400 was cut-off for positive for serovars Hardjo and Pomona in Farms A, B and F as they vaccinate with the 7 in 1 leptospiral vaccine. Figure 2 Age distribution of cattle with active infection with serovar Hardjo (MAT titre > 400) in the farms surveyed. Seroprevalence of maiden heifers (<2 years) is significantly lower than that of older cattle (p < 0.05). Discussion Serovar Hardjo (53.7%) is the major serovar present in the cattle, followed by Szwajizak (30.2%), Tarassovi (15.5%), Medanensis (12.8%) and Pomoma (8.4%). Serovars Szwajizak and Medanensis are

5 believed to demonstrate cross reactivity with serovar Hardjo (Black, et al. 2001) as they possess the same agglutinating epitopes (Faine, et al. 1999). This study has demonstrated serovar Hardjo infection is endemic in the cattle herd with > 30% in Papua New Guinea and may be responsible for the decrease reproductive efficiency in the cattle. The high seroprevalence of Hardjo in the non-vaccinated farms did not differ markedly from the vaccinated farms (Figure 1) indicating there is ongoing active infection of Hardjo in the farms. The practice of buying cattle from small holders can contribute to this as Hardjo is shed in the urine of older cattle who are chronic carriers. Control program based on changes in herd management can assist in preventing the increase within herd transmission. This was noticed in Farm B where a selected group of young heifers after weaning were separated from the dams and transported to a farm on a different location. Consequently, the chance for these heifers to be infected by older cattle is reduced. The infection of Hardjo in this group was very low (Figure 2). To eliminate infection from this group strict quarantine should follow after the cessation of vaccination. The practice of sharing a bull for mating should be restricted to prevent any transmission of leptospires (Ellis, et al. 1986). This controlled program and annual vaccination is to continue in the whole herd after five years there should be a significant reduction in the number of Hardjo infection in older cattle. A new serovar is moving into and infecting the cattle population which is Tarassovi. Pigs are the usual maintenance host for this serovar and are known to excrete the organism in their urine (Davos, 1977). Antibodies to serovar Tarassovi (15.8%) are also relatively common in the cattle population compared to Pomona (2.9%). Farms with high seroprevalence of Tarassovi have close association with pigs. Farm C had a piggery on the cattle property and runoffs from urine are washed into a small stream that runs through the paddock. This could be a possible route where Tarassovi is transmitted. Farm E is a local farm adjacent to a village and cattle grazed on open grass would come into contact with pigs, dogs and rodents which can be infected with Tarassovi. The infection in Farm A could be due to buying infected cattle from outlying villages. An earlier paper in Papua New Guinea reported the isolation of leptospires belonging to the serogroup Tarassovi from a bandicoot, Echymipera kalubu (Morahan, 1971). Bandicoots could be a possible source of transmission of Tarassovi to cattle as rodents and small mammals are the major host for a range of pathogenic serovars (Emanuel, et al. 1964; Glazebrook, et al. 1978; Kositanont, et al. 2003). Whether this is the case in Papua New Guinea is not known. Conclusion In conclusion the main leptospiral serovar infecting cattle is Hardjo and cattle are a potential zoonotic reservoir for serovars Hardjo, Tarassovi and Pomona in Papua New Guinea. Acknowledgements This study is supported by ACIAR project No. ASI/2001/054. The author acknowledges the assistance and support provided by the Papua New Guinea National Agriculture and Quarantine Inspection Authority (NAQIA) veterinary field staff and the management of the farms in collaborating in the survey. The author would like to thank Meegan Symonds and Michael Dohnt at the WHO/FAO/OIE Collaborating Centre of Reference and Research on Leptospirosis in Brisbane for performing the MAT and the isolation work.

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